Isolation of dna molecules

ABSTRACT

The present invention relates to a process for separating nucleic acid molecules, preferably open circular and super-coiled plasmid DNA and RNA molecules from each other, comprising the steps of providing a solution comprising the molecules; adsorbing the molecules to adsorbing groups on a carrier; and optionally washing the column with a suitable solution. The present process is especially suitable for large-scale isolation of supercoiled ccc DNA to be used in gene therapy.

TECHNICAL FIELD

[0001] The present invention relates to a process for separating nucleicacid molecules, such as plasmid DNA in a solution. More specifically,the present process is based on separation of oc (open circular) DNA andsupercoiled ccc (covalently closed circular) DNA as well as ribonucleicacids and other deoxyribonucleic acids from each other and othercomponents present in a solution.

BACKGROUND

[0002] The development of gene therapy and DNA vaccines has increasedthe demand for highly purified gene vectors such as plasmid DNA. Theproblem with the purification of supercoiled plasmid DNA is tocompletely remove other cell components such as host proteins,endotoxins, chromosomal DNA, RNA, open circular and nicked forms ofplasmid DNA.

[0003] Different chromatographic methods have been used for plasmid DNApurification, such as size exclusion chromatography, or gel filtration,hydroxyapatite, ion ex-change chromatography, reversed phasechromatography and hydrophobic interaction chromatography. Most of themethods lack the possibility to separate super-coiled plasmid DNA fromother forms of the plasmid. Many of the available methods also use RNaseto hydrolyse RNA in the cleared lysate before applying the sample to thechromatographic column. The usage of RNase is not recommendable in thepreparation of plasmid DNA that is intended for human use.

[0004] Ion exchange chromatography is the most commonly usedchromatography method. Plasmid DNA, chromosomal DNA and RNA all bind toanion exchangers as they have similar charge properties. Hydrophobicinteraction chromatography has also been used, however, the plasmid DNAdo not bind and was eluting in the flowthrough.

SUMMARY OF THE INVENTION

[0005] The object of the present invention is to provide a process forisolation of super-coiled plasmid DNA that avoids one or more of theabove-discussed drawbacks. Thus, one object of the present invention isto provide a process for isolation of nucleic acid molecules on amatrix, which is efficient. The object of the invention is morespecifically obtained by the process as defined in the appended claims,and by the matrix of the appended claims.

DEFINITIONS

[0006] The term “nucleic acid molecule” is understood herein to includelarge molecules and molecule aggregates, such as open circular plasmidDNA, supercoiled plasmid DNA and other DNA (e.g. genomic DNA) as well asRNA, such as mRNA, tRNA and sRNA.

[0007] The term “eluent” is used herein with its conventional meaning inchromatography, i.e. a solution capable of perturbing the interactionbetween the solid phase (adsorbent matrix) and product (nucleic acidmolecule/s) and promoting selective disassociation of the product fromthe solid phase.

[0008] It is to be understood that any term used in the presentspecification, but not specifically defined herein, is to be construedin accordance with the general meaning understood by those skilled inthe present technical field.

BRIEF DESCRIPTION OF THE DRAWINGS

[0009]FIG. 1 shows examples of aryl-S compounds reacted with Sepharose 6Fast Flow, which compounds do possess separation properties.

[0010]FIG. 2 shows the two chromatograms obtained after loadingrespectively 130 and 150 ml of cleared lysate on a Sephacryl S-500 HR,run in 2M ammonium sulphate. The insert shows the agarose gelelectrophoresis analysis of selected fractions as well as an aliquot ofthe starting material.

[0011]FIGS. 3A, B and C illustrate the results obtained afterchromatography on Sepharose 6 Fast Flow with a number of differentaryl-S ligands, as indicated on the chromatograms. Inserts show agarosegel electrophoresis analysis of selected fractions.

[0012]FIG. 4 illustrates results obtained after chromatography onSepharose 6 Fast Flow with a number of different aryl-S ligands, asindicated on the chromatograms.

[0013]FIG. 5 presents the chromatogram gained after loading the sample(prepurified by gel filtration) on Pyridyl-S Sepharose 6 Fast Flow underhigh conductivity conditions (>240 mS/cm).

[0014]FIG. 6 shows the results obtained after performing chromatographyof cleared lysate on Pyridyl-S Sepharose 6 Fast Flow in a XK16/15column.

[0015]FIG. 7 is a chromatogram using demonstrating the use of Na₂SO₄during adsorption of plasmid DNA to an S-aryl ligand according to theiunvention.

DETAILED DESCRIPTION OF THE INVENTION

[0016] In a first aspect, the present invention relates to a process forseparating nucleic acid molecules from a solution, comprising the stepsof

[0017] (a) subjecting a mixture of nucleic acid molecules to a matrix ofa carrier surface provided with an S-aryl ligand;

[0018] (b) subjecting the nucleic acid molecules to an elution step;

[0019] (c) isolating the different fractions containing the differentnucleic acid molecules; and

[0020] optionally washing the carrier with a suitable solution.

[0021] Thus, during step (a), the nucleic acid molecules are allowed toadsorb to the S-aryl ligands. The washing is performed after theadsorption but before the elution, as is well known in the art, in orderto remove retained undesired material. Naturally, the present processcan also be used in cases where nucleic acid is an undesired componentof a solution, i.e. to provide a soltion purified from nucleic acid. Inthat case, elution of nucleic acid molecules is performed forregeneration of the column. Accordingly, a method for purification of asolution according to the invention does not necessarily include a stepof elution.

[0022] In one embodiment, the process according to the invention is anisolation of nucleic acid molecules expressed in cells, and,consequently, it also comprises a first step of disintegrating the cellsto provide the solution comprising nucleic acid molecules. Suchdisintegration is performed e.g. by lysis, such as alkaline lysis,according to standard protocols (see e.g. Maniatis, T, Fritsch, E. F.and Sambrook, J. (1982) Molecular Cloning: A Laboratory Manual, ColdSpring Harbour Laboratory Press, Cold Spring Harbour, N.Y.).

[0023] In another embodiment, the present process comprises the furtherstep of eluting the supercoiled plasmid DNA molecules by contacting asuitable eluent with said matrix. Thus, the elution step can beperformed as a dynamic or batch procedure. Elution is convenientlyperformed according to well-known principles, such as by a gradient ofdecreasing conductivity as is also illustrated in the experimental partbelow.

[0024] Thus, the process according to the invention is utilised e.g. forpurification of nucleic acids for use in gene therapy, DNA vaccines andlaboratory studies related to gene therapy. In an advantageousembodiment, the present process will provide isolated supercoiledplasmid DNA of acceptable gene therapy grade. More specifically, it ispredicted that in a near future, there will be an increasing demand ofplasmid DNA, in large quantities for use in gene therapy as carriers orvectors of genetic material. As mentioned above, the previouslydescribed methods for isolation of such carriers have not beensatisfactory to this end, and the process according to the presentinvention is thus the first to enable large scale processing of nucleicacid molecules for medical and diagnostic use, in particular separatingoc plasmid DNA and ccc plasmid DNA. An additional advantage with thepresent method is that it is conveniently adapted to automation. Forexample, automation on ÄKTA™explorer (Amersham Pharmacia Biotech AB,Uppsala, Sweden) has been shown to result in high amounts of homogeneousplasmid DNA, more specifically in more than 98% supercoiled plasmid DNA.

[0025] Plasmids isolated in accordance with the invention can be of anyorigin. Most commonly, microorganisms like bacteria, such as E. coli,are used for culturing the plasmids, but the use of host cells is notlimited and can be prokaryotic or eukaryotic cells. The host cellsharbouring the plasmid can be cultivated in a number of ways well knownin the art, e.g. in incubator, bioreactor, fermentor etc. The plasmidisolated according to the invention can be of virtually any size, e.g.in the range of about 1 kb up to about 20 kb. As an upper limit, theisolation of cosmids and artificial chromosomes is also encompassed, thesize of which may be up to about 50 kb and 500 kb, respectively.

[0026] Plasmids can be of a high copy number or low copy number and cancarry any gene, either genomic or synthetic, encoding protein or peptideof interest, from any source. The culturing of the host cells, as wellas the exploitation of the plasmid for gene therapy, is well known inthe state of the art.

[0027] After culturing the host cells containing the plasmid, the cellsare recovered by e.g. centrifugation or filtration. The cells can bestored, for example in a freezer, or processed immediately.

[0028] As mentioned above, when the plasmid DNA according to theinvention has been produced in a cell, lysis thereof is advantageouslyperformed by alkaline lysis. The lysate may then be treated with metalions, such as of divalent alkaline earth metal ions, to precipitateimpurities and specifically RNA and chromosomal DNA. When theprecipitated material has been removed, the solution can be applied tothe column. (For a detailed disclosure of metal ion precipitationmethods in this context, see e.g. WO9916869 in the name of AmershamPharmacia Biotech.)

[0029] In an advantageous embodiment, the matrix material used ispresent as column chromatography material, and the carrier material ontowhich the ligand is bound, is any suitable inorganic or organicmaterial. Inorganic materials are glass, silica, or other inertparticulate minerals Such matrices can be any matrix available in themarket. There are many commercial products available based on differentresins or polymer, e.g. agarose or cross-linked agarose (such asSepharose™, Amersham Pharmacia Biotech), dextran (such as Sephadex™,Amersham Pharmacia Biotech), polystyrene/divinylbenzene (MonoBeads™,SOURCES, Amersham Pharmacia Biotech), coated polystyrene, acrylicpolymer, dextran acrylic polymer (Sephacryl™, Amersham PharmaciaBiotech), vinylic grafted polymer, or vinylic polymer, different silicabased resins such as silica-dextran, silica-acrylic polymer andsilica-polyethyleneimine.

[0030] The present process may be performed with the matrix as anexpanded bed, as a packed bed or in a batch mode. In packed bedadsorption, the adsorbent is packed in a chromatographic column and allsolutions used during a purification process flow through the column inthe same direction. In expanded bed adsorption however, the adsorbent isexpanded and equilibrated by applying a liquid flow through the column.A stable fluidized expanded bed is formed when there is a balancebetween particle sedimentation or rising velocity and the flow velocityduring application of the sample and washing steps. In the elution step,the adsorbent is precipitated and behaves like a packed bed adsorbent.

[0031] The ligand attached to the carrier material to form the matrix ofthe invention can be a mercapto-pyridine, mercaptoalkylpyridine, wherethe mercapto- and mercaptoalkyl groups are attached in ortho, and metaposition respectively, in relation to the pyridyl-nitrogen. Hereby it isthe mercapto-group that is attached to the carrier material via athioether binding. Further, aryl groups forming part of the ligand arephenyl, benzyl, toluyl, phenethyl, naphtyl, imidazolyl, pyrazolyl,pyrazinyl, pyrimidinyl, pyridazinyl, piperidinyl, morpholinyl,piperazinyl, indolyl, quinolinyl, purinyl. Further substituents can alsobe added on the aromatic ring. By providing the present S-aryl ligandswith additional substituents, a large range of different separationmedia can be designed in order to obtain desired binding and elutioncharacteristics. For example, it may be desired to shift the elutionprofile in order to allow use of a less concentrated eluent for thedesorption step. The substituents can for example be one or more aminegroups, nitro groups, ether groups, thiol groups and/or halogens, suchas fluorine. These additional substituents can also comprise furthercarbon atoms, as desired. Also, as the skilled in this field willrealise, carbon atoms can be exchanged for heteroatoms in the abovediscussed ring structures. It is to be understood herein that the term“S-aryl ligand” comprises a large range of compounds that can besubstituted to a desired extent, some of which will be exemplified belowin FIG. 1. The ligand density is 10-500 μmole/mL carrier, preferably inthe range 10-100 μmole/mL carrier.

[0032] The eluent used in the present invention is pH neutral(preferably a pH of 6.5 to 8.5) eluent, preferably an ammonium sulphatesolution having a concentration of 0.5 to 4 M, preferably 1.5 to 2.0 Mat which oc DNA is not bound in contrast to the ccc form. After loadingof the complete sample to the column, bound ccc plasmid DNA cansubsequently be eluted from the column by using decreasing ammoniumsulphate concentrations. RNA molecules can be eluted from the column byusing even lower concentrations of ammonium sulphate.

[0033] In a second aspect, the invention relates to matrices containinga mercapto-aryl moiety, which matrices selectively separates nucleicacid plasmid molecules, such as oc DNA, ccc DNA, and RNA molecules.Hereby, the mercapto-group may be substituted directly onto the arylgroup, or via an alkylene chain having 1 to 7 carbon atoms. Inlaboratory tests made it was shown that neither4(2-mercaptoethyl)pyridine, or 2-(2-oxoethyl)pyridine had the ability ofseparating ocDNA and cccDNA.

[0034] In one embodiment, the matrix particles are of a mean size in therange of about 10-300 μm, e.g. within a range of 10-20, 20-50, 50-100,100-200 or 200-300 μm. However, the particles can advantageously beprepared in any size for which commercially available sieve equipment isexist, such as 250, 212, 180, 150, 125, 106, 90, 75, 63, 45, 37, 30, 25,20, 15 μm.

[0035] In one embodiment, which is especially advantageous for theseparation of nanoparticles, such as plasmids or virus, the presentmethod will use a matrix comprised of one or more of the above discussedS-aryl compounds as ligands coupled to superporous particles. Theaverage superpore diameter of the superporous matrix particles used inthe present embodiment will be at least about 4 μm, such as about 5-10or about 10-20 μm, and maybe of a value of up to about 25 μm, such asabout 20-30 or even about 30-40 μm. However, in the present context, itis to be understood that the term “superporous” relates to particleswherein the pores are large enough so as to be an essential part of thestructure of the particles, i.e. to penetrate the particles to a muchdeeper extent than conventional particles having a porous surface layerbut a central portion which is essentially solid. In one especiallyadvantageous embodiment, the adsorption step is run under dynamicconditions. Accordingly, the superporous particles used in thisembodiment are to a substantial portion penetrated with pores, whilethey are still designed to be sufficiently rigid to keep their originalappearance, i.e. not to collapse, during a dynamic flow procedure.

DETAILED DESCRIPTION OF THE DRAWINGS

[0036]FIG. 1 shows examples of a variety of aryl-S compounds reactedwith Sepharose 6 Fast Flow. As appears from this drawing, the aryl-Scompounds can be provided with different substituents, each of which canresult in binding and elution properties that may prove advantageous fordifferent purposes.

[0037]FIG. 2 shows the results obtained after a group separation ofplasmid DNA versus RNA of cleared lysate on Sephacryl S-500 HR in a XK50/30 column with a bed height of 20 cm. The column is equilibrated in 2M (NH₄)₂SO₄ in 25 mM Tris, pH 7.9 with a conductivity of 216 mS/cm and130 respectively 150 ml cleared lysate is loaded at a flow rate of 30cm/hr. Once the sample is applied to the column, the flow rate isincreased to 60 cm/hr, and the void volume is collected. After totalelution of all sample and re-equilibration of the column, the nextsample is loaded. Analysis of selected fractions on a 1% agarose gelelectrophoresis shows the presence of open circular and supercoiledplasmid DNA together with RNA in the cleared lysate preparation. In theexcluded volume of the gel filtration, no presence of RNA can bedetected. However, most of both open circular and supercoiled plasmidDNA can be found in these fractions. The excluded volume is collectedand used in subsequent chromatography steps.

[0038]FIG. 3A depicts the chromatogram acquired after loading theplasmid DNA sample on a Pyridyl-S Sepharose 6 Fast Flow column andeluting with H₂O-gradient. The pyridyl-S ligand is depicted in theupper-right corner. Pyridyl-S Sepharose 6FF is equilibrated with 2 M(NH₄)₂SO₄ in 25 mM Tris, pH 7.9 (216 mS/cm) in a 4.6/15 PEEK-columnafter which 20 ml of sample obtained after gel filtration chromatographyis loaded on the Pyridyl-S Sepharose 6FF at a flow rate of 45 cm/hr.After washing off all unbound material with equilibration buffer, thesupercoiled plasmid DNA is eluted by decreasing the conductivity byapplying a gradient over 2 column volumes with H₂O. One peak elutes whenthe conductivity reaches 212 mS/cm. Once the total gradient has beenestablished, the column is re-equilibrated with 2 M (NH₄)₂SO₄ in 25 mMTris, pH 7.9 (216 mS/cm). Selected fractions from void volume andelution peak are analyzed for the presence of the different forms ofplasmid DNA on a 1% agarose gel electrophoresis. The insert shows thatunder these conditions, most of the open circular plasmid DNA does notbind to the Pyridyl-S Sepharose 6 Fast Flow, while the supercoiled DNAbinds to the media and can be eluted by lowering the conductivity toless than 212 mS/cm. The last lane on the agarose gel represents a fivetimes dilution of the elution peak containing the supercoiled plasmidDNA.

[0039]FIG. 3B represents the results obtained after loading the plasmidDNA sample obtained after gel filtration on the Sephacryl S-500 HRcolumn on a 2-mercaptoethylpyridine Sepharose 6 Fast Flow column. Theligand used in this experiment is depicted in the upper-right corner ofthe chromatogram. After equilibration of the column with 20 ml of thesample obtained in the experiment illustrated in FIG. 1 is loaded on thecolumn at 45 cm/hr. After 2 column volumes of washing s buffer, thesupercoiled plasmid DNA is eluted from the column by decreasing theconductivity with a gradient of H₂). Once the conductivity reaches avalue of 209 mS/cm, the bound material elutes from the column. Asillustrated in the 1% agarose gel electrophoresis, this elution peakcontains mainly supercoiled plasmid DNA while the open circular plasmidDNA can be found in the void volume, and thus does not bind to the mediaunder these settings.

[0040]FIG. 3C shows the chromatogram obtained with the ligand depictedin the upper-right corner of the figure. The phenyl-S Sepharose 6 FastFlow column is equilibrated with 2 M (NH₄)₂SO₄ in 25 mM Tris, pH 7.9(216 mS/cm) before loading 20 ml of the void volume of the gelfiltration on the media at a flow rate of 45 cm/hr. Unbound material iswashed off with 2 M NH₄)₂SO₄ in 25 mM Tris, pH 7.9 (216 mS/cm), andbound material is eluted by lowering the conductivity with aH₂O-gradient. At a conductivity of 214 mS/cm, the material starts toelute from the media. Analysis on a 1% agarose gel electrophoresis showsthe presence of only open circular plasmid DNA in the flow through.Supercoiled plasmid DNA binds to the column and is detected in theelution peak.

[0041]FIG. 4 shows a number of chromatograms obtained with some of thedifferent ligands depicted in FIG. 1. For every chromatogram, the ligandis presented in the upper-right corner of the figure. The aryl-SSepharose 6 Fast Flow column is equilibrated with 2 M (NH₄)₂SO₄ in 25 mMTris, pH 7.9 (220 mS/cm) before loading 20 ml of the void volume of thegel filtration on the media at a flow rate of 45 cm/hr. Bound materialis eluted by decreasing the conductivity with a gradient to pure H₂ 0.By comparing the different chromatograms, it becomes clear that bychanging the ligand properties, the binding and elution conditions forplasmid DNA can be modified.

[0042]FIG. 5 illustrates the results obtained after loading the plasmidDNA sample on Pyridyl-S Sepharose 6 Fast Flow under high conductivityconditions. The Pyridyl-S Sepharose 6 Fast Flow is equilibrated with 2.4M (NH₄)₂SO₄ in 25 mM Tris, pH 7.9 (240 mS/cm). The sample obtained aftergel filtration on the Sephacryl S 500 HR is adjusted to 240 mS/cm beforeloading 20 ml on the Pyridyl-S Sepharose 6FF at 45 cm/hr. Unboundmaterial is washed out using the same buffer, and elution is started bydecreasing the conductivity with a H₂O-gradient. Two elution peaks aredetected under these conditions, and selected samples are analyzed on a1% agarose gel electrophoresis. Under these circumstances, both opencircular and supercoiled plasmid DNA are found to bind the Pyridyl-SSepharose 6 FF, but both tend to elute at different conductivity-valuesand thus factions enriched in either open circular or supercoiled DNAcan be acquired.

[0043]FIG. 6 shows the results obtained after performing chromatographyof cleared lysate on Pyridyl-S Sepharose 6 Fast Flow in a XK16/15column. The Pyridyl-S Sepharose 6 Fast Flow column is equilibrated with2 M (NH₄)₂SO₄ in 25 mM Tris, pH 7.5 (224 mS/cm) after which 150 ml ofcleared lysate is loaded at a flow rate of 90 cm/hr. Unbound material iswashed out, and the bound material is eluted by decreasing theconductivity by applying a gradient with water over the column. Opencircular plasmid DNA does not bind to the matrix, while both supercoiledplasmid DNA and RNA do bind with the Pyridyl-S Sepharose 6 FF. Both canhowever be eluted at different conductivities, where supercoiled plasmidDNA starts eluting from the column at 211 mS/cm, while RNA only elutesin a broad peak once the conductivity is below 170 mS/cm.

[0044]FIG. 7 illustrates how plasmid DNA is loaded on a column accordingto the invention preconditioned in 3.0 M Na₂SO₄, 10 mM EDTA, 100 mMTris-HCl, pH 7.0 and eluted with a gradient to 1M NaCl.

[0045] Experimental Part

[0046] Below, the present invention will be described by way of examplesprovided only as an illustration and not to be construed as limiting thescope of the invention as defined by the appended claims in any way. Allreferences included below or else-where in the present application arehereby included herein by reference.

[0047] Preparation of Ligand Comprising Agarose Beads

[0048] Sepharose 6 Fast Flow (Amersham Pharmacia Biotech) was activatedwith epoxy groups according to Hermanson et al [Immobilized AffinityLigand Techniques, Academic Press (1992), p. 118]. The ligand densitywas approximately 50 μmol epoxy/mL carrier. The aryl-S reagents (SeeFIG. 1), 50 μmol/mL carrier, were reacted with the epoxyactivatedSepharose at pH 10.5. The reactions were performed at 45° C. undernitrogen atmosphere over night. After the reaction, the gel was cooledand washed with acetone and finally water. The ligand concentrationobtained were in the range of 30-50 μmol/mL carrier.

[0049] Cell Culture

[0050] An inoculum of E. Coli TG1 cells containing a pUC19 plasmid withJV4-insert are grown in 2YT medium to a OD of 4, after which 40 ml istransferred to a 10 liter culture in a Biostat ED reactor. At an OD of13.6, the cells are harvested, centrifuged for 40 minutes at 4200 rpm ina Sorvall RC12BP rotor and the cell pellet is stored at −70° C.

[0051] Alkaline Lysis

[0052] 25 g of bacteria are resuspended in 50 ml of ice-cold S buffer(61 mM glucose, 10 mM Tris, 50 mM EDTA pH 8.0). Another 130 ml of bufferS is added. Under constant gentle stirring, 390 ml of buffer P2 (0.2 MNaOH, 1% SDS) is added. After 10 minutes of gentle stirring at roomtemperature to assure complete mixing, 293 ml ice-cold buffer P3 (3Mpotassium acetate, pH 5.5) is added. Under gentle stirring for 20minutes, the solution is incubated on ice before storing overnight at 4°C. The next day, the mixture is centrifuged for 30 minutes at 10.000 rpmat 4° C. in a GSA rotor and the supernatant is filtered through filterpaper to obtain cleared lysate.

[0053] Sample Preparation

[0054] Cleared lysate has been processed as follows:

[0055] a) adjusted to 2M ammonium sulphate by addition of equal amounts(volumes) of 4M ammonium sulphate, or preferentially

[0056] b) sample prepurification by size exclusion (group separation andbuffer exchange) on Sephacryl™ S-500 HR (Amersham Pharmacia Biotech),pre-equilibrated and run in 2M ammonium sulphate. A Sephacryl S-500 HR(XK 50/30 column, 20 cm bed height) is equilibrated with 2 M (NH₄)₂SO₄in 25 mM Tris, pH 7.9. Up to 0.4 CV of cleared lysate is loaded on thecolumn at 30 cm/hr. Once all sample is loaded on the column, the elutionspeed is adjusted to 60 cm/hr, and the flow through containing plasmidDNA is collected for further experiments (see FIG. 2).

[0057] Chromatography

[0058] Several aryl-S Sepharose 6 Fast Flow media are packed in 4.6/15PEEK- or XK 16/15 glass-columns (15 cm bed height) at 140 cm/hr and allcolumns are equilibrated in 2 M (NH₄)₂SO₄ in 25 mM Tris, pH 7.9resulting in a conductivity of more then 215 mS/cm (corrected for 25° C.temperature) at 45 cm/hr. 20-150 ml of cleared lysate preparations(according to a) or b), see above) is then loaded to the column at thesame flow rate. After a wash of 2 column volumes, the bound material iseluted from the media with a gradient to H₂O over 10 column volumes.During the run, absorption at 260 nm is recorded. Different fractionsare collected and a number of them are analysed on a 1% agarose gelelectrophoresis stained by ethidium bromide and visualised by UV (seeFIGS. 3 A-C, FIG. 4, FIG. 5 and FIG. 6).

1. A process for separating nucleic acid molecules from a solution, comprising the steps of (a) subjecting a mixture of nucleic acid molecules to a matrix of a carrier surface provided with an S-aryl ligand; (b) subjecting the nucleic acid molecules to an elution step; (c) isolating the different fractions containing the different nucleic acid molecules; and (d) optionally washing the carrier with a suitable solution.
 2. A process according to claim 1, which comprises a first step of disintegrating cells, wherein the nucleic acid molecules are expressed, to provide the solution comprising nucleic acid molecules, which disintegration is a lysis.
 3. A process according to claim 1 or 2, which comprises a further step of eluting the nucleic acid molecules by contacting a suitable eluent.
 4. A process according to any one of claims 1-3, wherein the carrier is an inorganic matrix.
 5. A process according to claim 4, wherein the carrier is glass.
 6. A process according to claim 4, wherein the carrier is a zeolite.
 7. A process according to claim 4, wherein the carrier is a silica.
 8. A process according to any one of claims 1-3, wherein the carrier is an organic matrix.
 9. A process according to claim 8, wherein the carrier is a resin or polymer.
 10. A process according to claim 9, wherein the carrier is agarose or cross-linked agarose, dextran, polystyrene/divinylbenzene, coated polystyrene, acrylic polymer, dextran acrylic polymer, vinylic grafted polymer, or vinylic polymer.
 11. A process according to claim 9, wherein the carrier is different silica based resins in the form of silica-dextran, silica-acrylic polymer or silica-polyethyleneimine.
 12. A process according to one or more of the preceding claims, wherein the carrier is present as an active surface to which the nucleic acid molecule mixture is subjected.
 13. A process according to one or more of the preceding claims, wherein the carrier is present as a membrane which comes in contact with the nucleic acid molecule mixture.
 14. A process according to one or more of the preceding claims, wherein the carrier is present as filter aid which comes in contact with the nucleic acid molecule mixture.
 15. A process according to one or more of the preceding claims, wherein the carrier is present as a chromatography matrix in a column through which the nucleic acid molecule mixture is passed.
 16. A process according to one or more of the preceding claims, wherein the carrier is present as a sponge material to which the nucleic acid molecule mixture is subjected.
 17. A process according to one or more of claims 1-16, wherein the S-aryl ligand is a pyridyl-S ligand having the S-moiety bound in 2 (ortho) position to the pyridyl nitrogen atom.
 18. A process according to one or more of claims 1-16, wherein the S-aryl ligand is a pyridyl-S ligand having the S-moiety bound in 3 (meta) position to the pyridyl nitrogen atom.
 19. A process according to claim 17, wherein the S-aryl ligand is a pyridyl-S ligand having the S-moiety bound in 2 (ortho) position to the pyridyl nitrogen atom and separated to the pyridyl moiety via an alkylene chain having 1 to 7 carbon atoms, preferably 1 to 4 carbon atoms.
 20. A process according to claim 18, wherein the S-aryl ligand is a pyridyl-S ligand having the S-moiety bound in 3 (meta) position to the pyridyl nitrogen atom and separated to the pyridyl moiety via an alkylene chain having 1 to 7 carbon atoms, preferably 1 to 4 carbon atoms.
 21. A process according to one or more of claims 1-16, wherein the S-aryl ligand is a phenyl-S ligand.
 22. A process according to claim 21, wherein the S-aryl ligand is a phenyl-S ligand having the phenyl moiety separated to the S-group via an alkylene chain having 1 to 7 carbon atoms, preferably 1 to 4 carbon atoms.
 23. A process according to the preceding claims, wherein the nucleic acid molecules comprises oc plasmid DNA, ccc plasmid DNA, genomic DNA, and RNA.
 24. A matrix for separating for separating nucleic acid molecules, characterized in that it comprises an aryl-S ligand bound to a carrier to form a matrix, with the provisio that the ligand is not 2-(mercaptomethyl)pyridine, 4-(mercaptomethyl)pyridine, 2-(2-mercaptoethyl)pyridine, 4-(2-mercaptoethyl)pyridine, 4-nitro-1-mercaptomethyl-benzene, 3,4-dinitro-1-mercaptomethylbenzene, 2-mercaptomethylquinoline, mercaptoethylimidazoline, and 6-mercaptomethyluraciline.
 25. A matrix according to claim 24, wherein the S-aryl ligand is a pyridyl-S ligand having the S-moiety bound in 2 (ortho) position to the pyridyl nitrogen atom and optionally separated to the pyridyl moiety via an alkylene chain having 1 to 7 carbon atoms, preferably 1 to 4 carbon atoms.
 26. A matrix according to claim 24, wherein the S-aryl ligand is a pyridyl-S ligand having the S-moiety bound in 3 (meta) position to the pyridyl nitrogen atom and optionally separated to the pyridyl moiety via an alkylene chain having 1 to 7 carbon atoms, preferably 1 to 4 carbon atoms.
 27. A matrix according to claim 24, wherein the S-aryl ligand is a phenyl-S ligand, optionally having the S-group separated to the phenyl moiety via an alkylene chain having 1 to 7 carbon atoms, preferably 1 to 4 carbon atoms.
 28. A column for separation and purification of nucleic acid plasmid molecules, characterized in that it comprises a matrix onto which an aryl-S ligand is linked with the provisio that the ligand is not 2-(mercaptomethyl)pyridine, 4-(mercaptomethyl)pyridine, 2-(2-mercapto-ethyl)pyridine, 4-(2-mercaptoethyl)pyridine, 4-nitro-1-mercaptomethyl-benzene, 3,4-dinitro-1-mercaptomethylbenzene, 2-mercaptomethylquinoline, mercaptoethylimidazoline, and 6-mercaptomethyluraciline.
 29. A column according to claim 28, wherein the S-aryl ligand is a pyridyl-S ligand having the S-moiety bound in 2 (ortho) position to the pyridyl nitrogen atom and optionally separated to the pyridyl moiety via an alkylene chain having 1 to 7 carbon atoms, preferably 1 to 4 carbon atoms. 30 A column according to claim 28, wherein the S-aryl ligand is a pyridyl-S ligand having the S-moiety bound in 3 (meta) position to the pyridyl nitrogen atom and optionally separated to the pyridyl moiety via an alkylene chain having 1 to 7 carbon atoms, preferably 1 to 4 carbon atoms. 31 A column according to claim 28, wherein the S-aryl ligand is a phenyl-S ligand, optionally having the S-group separated to the phenyl moiety via an alkylene chain having 1 to 7 carbon atoms, preferably 1 to 4 carbon atoms. 